Surface-Mediated DNA Hybridization: Effects of DNA Conformation

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Surface-Mediated DNA Hybridization: Effects of DNA Conformation, Surface Chemistry, and Electrostatics Jeremiah Traeger, and Daniel K Schwartz Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02675 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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Surface-Mediated DNA Hybridization: Effects of DNA Conformation, Surface Chemistry, and Electrostatics

Jeremiah C. Traeger and Daniel K. Schwartz* Department of Chemical and Biological Engineering University of Colorado Boulder, Boulder, Colorado 80309

*Address correspondence to [email protected]

ACS Paragon Plus Environment

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Abstract Single-molecule Förster Resonance Energy Transfer (FRET) was used to study the dynamic association of mobile donor-labeled ssDNA oligonucleotides (“target”) with covalently immobilized complementary acceptor-labeled ssDNA oligonucleotides (“probe”). While probetarget association events were resolved for all experiments, such FRET events were far more likely to occur in systems with complementarity and on hydrophobic, as compared to hydrophilic, surfaces. The distribution of donor-acceptor association-time intervals did not exhibit simple first-order kinetics, and when decomposed into a superposition of first-order processes, only a small fraction of events corresponded to a long-lived state that was presumed to represent true DNA hybridization, while the majority of association events were transient, representing non-specific associations or partial hybridization. The structure of the DNA target and probe affected both the stability of the hybridized state, as well as the likelihood that an association between the two led to hybridization. In particular, the likelihood of hybridization decreased for longer target strands and for targets with stem-loop secondary structure. The presence of oligonucleotide secondary structure reduced the stability of hybridization, while greater complementarity increased stability of the hybridized state. Interestingly, increased ionic strength (i.e. greater electrostatic screening) increased the probability of hybridization, but did not influence the lifetime of the hybridized state. Combined, these observations provide a nuanced view of surface-mediated DNA hybridization, where various factors independently influence the probability and stability of hybridization.


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Introduction DNA-based nanotechnologies such as sequencing1, amplification2, sequence detection3, and mapping4 often rely on a solid-liquid interface to perform their desired function. Many of these applications involve a nucleic acid “probe” that is covalently attached to the surface and comprises a nucleotide sequence that is designed to interact with a complementary nucleic acid “target” 5,6 that is dissolved in buffer and exposed to the surface.

Fig 1. Schematic of the kinetic pathway for surface-mediated DNA hybridization.

The general concept of surface-mediated hybridization is illustrated in Figure 1. In this paradigm, free oligonucleotide targets diffuse to the surface, adsorb nonspecifically, and then engage in two-dimensional “searching” behavior7, and potentially come into contact with an immobilized probe molecule8. It is believed that the initial DNA-DNA interactions are dominated by attractive π-π stacking interactions, which act over longer ranges than WatsonCrick base pairing9,10. If the oligonucleotides have complementary sequences, a hybridized region between two short complementary sequences may nucleate, and the chains may eventually anneal to form a DNA duplex11. If imperfect out-of-sequence base pairing occurs, the two strands may change conformation via “inchworm”-like movement until they arrive at the optimal dsDNA state12. While in the dsDNA state, thermal fluctuations may cause the duplex to open up into a “bubble”13, nucleating a melting event, where the chains dissociate.


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This prevailing model of DNA hybridization and melting, which involves the nucleation and growth of hybridization-regions and bubbles14 has been primarily explored in homogeneous solution, in the absence of vicinal surfaces, which have shown to modify the chemical and physical kinetics of nucleic acids15. Considering that a variety of technologies rely on interfacial behavior to perform their function, an improved understanding of the surface-mediated mechanisms of DNA hybridization and melting may lead to improved performance of applications that rely on DNA hybridization/melting in the near-surface environment.

Single-molecule (SM) microscopy provides important insights into molecular interactions at solid-liquid interfaces16,17. In particular, data involving detailed temporal trajectories of large numbers of individual molecules explicitly enumerate coupled elementary dynamic mechanisms that would not be resolved in a steady-state ensemble-averaging measurement. Total Internal Reflection Fluorescence Microscopy (TIRFM) facilitates the excitation of fluorescently labeled molecules near an interface, and intermolecular Fӧrster Resonance Energy Transfer (FRET) can be used to investigate dynamic interactions between molecules labeled with a donor or acceptor dye, respectively. Interestingly, previous SM-FRET studies of surface-mediated DNA associations have revealed complex association kinetics that cannot be reduced to a single simple first-order melting behavior8,18 These reports also suggest that even when DNA pairs are noncomplementary, they can associate into a transient state that is stable for a timescale on the order of hundreds of microseconds without hybridizing. This may be due to 𝜋 − 𝜋 stacking interactions19, which can stabilize the two strands nonspecifically, as well as transient out-ofsequence pairing involving the formation of temporary Watson-Crick “bonds”.


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DNA hybridization is often simplistically described as a cooperative process, where the “equilibrium” state of the system, single-stranded or double-stranded, depends on factors such as temperature, DNA concentration, and ionic strength. Thus, below the so-called equilibrium melting temperature, oligonucleotide pairs are much more likely to be found in a double-stranded state than a single-stranded state. However, like all such phenomena, this equilibrium is actively dynamic, and many experiments have identified and quantified the presence of dynamic binding/unbinding events, even at temperatures well below the melting temperature. SM-FRET, in particular, has in recent years allowed for the investigation of kinetics of this dynamic equilibrium, giving insight into denaturation dynamics on an molecular level that would not be apparent in bulk measurements.

For example, Liu et al.20 used single-molecule measurements of oligonucleotides in solution below the melting temperature, where FRET traces of complementary pairs indicated transient associations between strands. Association events between 8-nucleotide complementary strands exhibited characteristic lifetimes of 40-100 ms even at room temperature. DNA hairpins have been shown to be similarly dynamic, as shown by Chen et al.21 where the self-hybridization dynamics of surface-immobilized hairpin structures were probed using FRET. This study identified brief transient openings (on the order of tens of µs) well below the melting temperature. This suggests that while the presence of secondary structure may prove to be a barrier to hybridization between a target and a probe, nucleotides can be exposed on timescales sufficiently long to nucleate hybridization between a hairpin target and a complementary probe. Other studies in recent years for both complementary DNA strands22–24 as well as hairpins25 have demonstrated dynamic behavior for DNA denaturation, even below the melting point.


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The existence of surface-mediated transient non-specific associations will clearly influence the overall distribution of association events, and in turn will affect macroscopic phenomena, including the performance of devices that rely on hybridization. Here, SM-FRET is used to elucidate the molecular-level mechanisms of non-specific surface-mediated DNA associations that may compete with, and hinder or destabilize, specific hybridization.

Experimental Section Materials 3-Glycidoxypropyltrimethyoxysilane (95% pure) was obtained from Gelest; n-butylamine (99.5% pure) was obtained from Sigma-Aldrich; Micro 90 cationic detergent was obtained from International Product Corp. Methyl-(polyethylene glycol)4-thiol (98% pure) was obtained from Gelest. All other chemicals were Optima grade from Fisher Scientific. All chemicals were used as received without further purification or modification. Aqueous solutions were prepared with water purified to 18 MΩ ∙cm using a Millipore Milli-Q UV+ system. DNA sequences and their modifications are listed in Table 1. The target molecules dissolved in solution were oligonucleotides obtained from Invitrogen. For the fifteen-nucleotide (15nt) and control sequences, the 5’ end was modified with a Fluorescein dye via C6-Amino linker, where the fluorescein dye acts as the donor fluorophore. The 30-nucleotide (30nt) and hairpin target sequences were internally modified with a Fluorescein dye on a thymine base. The probe molecules (attached to the surface) were oligonucleotides with a C6-Amino linker on the 5’end, and they were obtained from Biosearch. The 15nt probe was labeled with Quasar 670 dye via C7-Amino linker on the 3’ end, where the Quasar 670 dye acts as the acceptor fluorophore. The


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30nt probe was internally modified with a Quasar 670 dye on a thymine base. DNA solutions were prepared in PBS solution at concentrations of 10-10 M to achieve low enough surface densities for single-molecule experiments. These were prepared using 7.4 pH 10× PBS solution, which was diluted to 5× or 1× PBS when the DNA strands were to be introduced at those PBS concentrations.

Table 1. DNA sequences employed in the single-molecule FRET experiments. Identical sequences are labeled accordingly and AC sequences that are complementary to a TG sequence have an asterisk. . Stem-loop strand sequences in bold are self-complementary. Probes are tagged with Quasar 670 dye at the asterisk. Targets are tagged with fluorescein dye at the asterisk. Probes were immobilized using a reactive amine on the 5’ end followed by a 6carbon spacer. Probe sequences

# Bases

30nt Probe

NH2-C6-5’-TGG TGT GTT GGT TGT T*TT GGG TGG TTT GTG -3’

15nt Probe

NH2-C6-5’-TGG TGT GTT GGT TGT -3’*

1

2

30 15

2

Target sequences 30nt Target

5’- CAC AAA CCA CCC AAT* ACA ACC AAC ACA CCA -3’

15nt Target

5’*- ACA ACC AAC ACA CCA -3’

Hairpin Target

5’-TGG TGT GTT GGT TGT AAT TT*A CAA CCA ACA CAC CA-3’

Control Target

5’*- GAT TAG TAG TTT GGC -3’

2*

1*

15

1* 1

30

1*

35 15

Surface Preparation Surface preparation was based on a previous method developed by Monserud8,26. 2” diameter fused silica wafers were first cleaned by rinsing sequentially with 2% Micro 90 detergent solution in water, deionized (DI) water, toluene, and 2-propanol, before drying with ultra-highpurity nitrogen. The cleaned wafers were then placed in piranha solution (3:1 sulfuric acid/hydrogen peroxide by volume) at ~70 oC for 1-2 hours to remove any organic contaminants


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bonded or physisorbed to the surface. Subsequently, wafers were further cleaned by Micro-90 surfactant solution, DI water, toluene, and 2-propanol and again rinsed by ultra-high purity nitrogen. They were treated with UV-ozone for 2 hours, which thoroughly removed any remaining organic contaminants. The wafers were then placed in a vacuum chamber with a 1:3:20 solution by volume of n-butylamine/3-glycidoxypropyltrimethoxysilane(GPTMS)/toluene for 16-20 hours at room temperature, where GPTMS was vapor deposited on the surface of the wafers. The formation of a GPTMS monolayer was confirmed by measurement of a water contact angle of ~55o. The GPTMS monolayer exhibited exposed epoxide rings, which reacted with the probes as well as the thiolated surface-modification reagents. The GPTMS-coated wafers were then removed from vacuum.

For DNA immobilization, 19 mg of anhydrous lithium hydroxide was added to 100 mL of dimethylformamide in a foil-wrapped beaker. For hydrophobic surfaces, 106 µL of 1dodecanethiol was added to the solution. For hydrophilic surfaces, 100 mg of methyl-PEG4-thiol was added instead. 1 mL of 1µM DNA probe molecules were then added to the solution. The beaker was placed on an orbital shaker and agitated at 150 RPM for 48 hours. The wafer was removed from the deposition mixture, and thoroughly rinsed with Micro-90 solution, Milli-Q water, toluene, and isopropanol, before drying with nitrogen and storage under vacuum conditions at room temperature. The alkane surface had a measured contact angle of ~65 o, while the OEG surface had a contact angle of ~20o.


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Microscopy A flow cell was cleaned using subsequent rinses of Micro-90 detergent solution, Milli-Q water, toluene, and isopropanol before drying with nitrogen. A prepared wafer with immobilized probe DNA was placed into the flow cell and mounted on a custom-built prism-based TIRF microscope (Nikon TE-2000 base, 60× water-immersion objective) held at 22 oC. The wafer was exposed to a solution of target oligonucleotides within the flow cell. A 491 DPSS laser was used as an excitation source incident through a hemispherical prism placed in contact with the back of the wafer, creating TIRF field with a decay length of ~118 nm. The intensity of illumination was set at 35 mW, which was sufficient to allow continuous movie-length observations while avoiding significant photobleaching on the time-scales of the association events. Fluorophores in solution diffused too rapidly to be resolved, so all objects that appeared within the image were adsorbed to the surface.

Movies were captured for 2 minutes at 100 ms per frame such that each movie contained 1,200 frames. 6-16 movies were taken per experimental condition to obtain sufficiently large data-sets. An Optosplit II image splitter (Cairn Research) was used to split the light through the objective via a 610 nm dichroic mirror to separate light from donor and acceptor fluorophores. The donor channel used a 525 nm filter with a 90% transmission width of 28 nm, while the acceptor channel used a 575 nm filter with a 40 nm 90% transmission width. Filters were from Semrock. The images in the two channels were captured with an EMCCD camera cooled to -92 oC from Photometrics. Before capturing movies, the channels were aligned using an alignment grid to within 1-2 pixels, to be further corrected in image processing. At the end of each experiment, 1.0 𝜇𝑚 fluorescent latex beads from Life Technologies were exposed to the surface, where they


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emitted fluorescence visible within both channels, permitting more precise channel alignment in post processing.27

Image Analysis Each object was identified using a disk matrix and a thresholding algorithm28. In each frame both channels were convolved with a disk matrix simulating the point spread function so that objects in the same position in each channel could be identified. The locations of the objects were identified by calculating the center of intensity. The intensities of all the pixels associated with each objects were integrated and subtracted from that object’s median background intensity to determine the fluorescence intensity. Objects within 3 pixels of each other in consecutive frames (equivalent to 681 nm) were tracked as the same object. Objects that appeared within 2 pixels of each other (454 nm) within separate channels were identified as the same object undergoing a FRET event. The position of the event was identified by the position of the object in the channel with the greatest signal-to-noise ratio. In previous work, to parameterize donor-acceptor energy transfer, the relative distance between objects was calculated using the following equation: 1

𝑑=

𝐹 6 (𝐹𝐷 ) 𝐴

(1)

While this parameter has a superficial similarity to the actual donor-acceptor distance (e.g. when multiplied by the Förster radius), here it is used simply as a dimensionless parameter, the socalled “relative distance”, that allows us to identify FRET states. This allows for the identification of FRET between fluorophores, indicating relative distances between DNA strands26, ends of polymers27, and the degree of unfolding in a protein29, among other applications. Specifically, the “state” of each object was determined using a threshold d-value dividing one state from another. While this process was simple and intuitive, it had the


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disadvantage of reducing data that was fundamentally two-dimensional (intensities in donor and acceptor channels) to a single ratio. We found that this could sometimes “hide” populations by projecting two populations onto one in the reduced-dimensional data space. In order to get a firmer grasp of the populations that existed within the data for each experiment, the donor intensity and the acceptor intensity for each object were plotted to create a two-dimensional heatmap, upon which each FRET population would appear as a peak or cluster, as shown in Figure 2.

In addition to the expected populations corresponding to high FRET efficiency (low donor-high acceptor) and low FRET efficiency (high donor-low acceptor), this analysis allowed us to identify a population of FRET artifacts with relatively low intensity in both channels, which would not have been apparent as a separate population in a one-dimensional analysis. We determined that nearly all of these latter trajectories involved objects that were brief (1-2 frames) and anomalously small in size (1-2 pixels) compared to the apparent size of the actual point spread function, and were therefore attributed to noise. Upon removal of all trajectories comprising objects less than three pixels in size, this population near the origin disappeared, indicating there were only two physical states: associated (high-FRET) and unassociated (lowFRET), which could be clearly distinguished using an optimal linear boundary within the twodimensional donor-acceptor intensity plane.


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Fig 2. A heat map representing the fluorescent intensity for each object in both donor and acceptor channels. Two populations of FRET states were observed, one with high-FRET and one with low-FRET, with no apparent intermediate states.

States, Lifetimes and Distributions For each experiment, 50,000 – 600,000 trajectories were captured, and the donor and acceptor emission intensities of each object in each frame were used to prepare a two-dimensional heatmap as shown in Figure 2. Within a given trajectory, each frame was identified as being either associated or unassociated. For each experiment, the fraction of trajectories that exhibited at least one association (i.e. FRET) event was calculated as fe, i.e. the “event fraction”, representing the probability that a given adsorbed target molecule would engage in a measurable interaction with an immobilized probe molecule. State lifetimes were determined by identifying the states of the objects within each trajectory, tallying the number of consecutive frames associated with each state, and multiplying by the acquisition time (100 ms). These were accumulated and assembled into a cumulative probability distribution of lifetimes within each state.


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In order to decompose the distributions into characteristic time scales, the probability distributions were modeled using an exponential mixture model 𝑃(𝑡) = 𝐴 ∑𝑖 𝑓𝑖 𝑒

−

𝑡 𝜏𝑖

(2)

where each exponential mode was associated with a first-order decay timescale (i) and an associated fraction (fi); the latter summed to unity. These were all multiplied by a constant less than one to account for the objects that were not resolvable due to extremely short surface lifetimes. In order to determine the optimal number of modes to fit to these data, a maximumentropy method analysis was performed. This transforms exponential timescales into Laplace space, ultimately producing a peak for each exponential decay mode30,31. We found that two exponential functions were appropriate to describe the data in all experiments, except for the control experiments which exhibited one broad peak.

Results DNA Systems: Probes and Targets In order to investigate the effect of strand length and target structure for hybridization, multiple DNA systems were analyzed as illustrated in Figure 3 (see Table 1 for specific sequences). The 15 base pair (“15bp”) system involved a 15nt probe DNA oligomer (composed of T and G nucleotides) and a complementary 15nt target DNA oligomer (composed of A and C nucleotides), which formed a 15bp dsDNA duplex when fully annealed. The “overhang” system employed the same 15nt surface-immobilized probe, but introduced a 30nt target oligomer in solution containing the same AC sequence as the 15nt target. When fully annealed, this resulted in a 15bp dsDNA duplex with a 15nt ssDNA overhang. The “hairpin” system again used the


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same 15nt immobilized probe in conjunction with a target in solution that was capable of selfhybridization into a stem-loop structure with a 15nt stem and a 5-base loop. The hairpin target could also hybridize to the probe, resulting in a 15bp dsDNA duplex with a 20-base overhang. The 30 base pair (“30bp”) system comprised a 30nt immobilized probe and a complementary 30-base target in solution. Because of the labeling strategy, when fully annealed, there was a single mismatch in the form of two “paired” thymine bases, because the 30nt ssDNA strands required these bases for the internal modification. Thus, the annealed structure had 29 complementary base pairs with a single internal mismatch. Finally, the “control” system involved a 15nt immobilized probe with a non-complementary 15nt target in solution.

Fig. 3. A schematic representation of the probe-target systems investigated. The target strands were functionalized with fluorescein, which acted as the donor fluorophore. The probe strands covalently attached to the surface were functionalized with Quasar 670, the acceptor fluorophore.

Event Fractions As described above, following adsorption each target molecule engages in “searching” behavior that may result in a significant encounter with an immobilized probe, resulting in a FRET event that lasts long enough to be resolved and detected. The fraction of adsorbed target molecules that


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successfully resulted in at least one FRET event was defined as the event fraction, fe, as shown in Figure 4 and Table 2. Notably, the non-complementary control had an extremely small event fraction of only 0.06, suggesting that in the absence of complementarity, most associations were too transient to result in detectable FRET. For systems with some degree of complementarity, the event fractions were much larger, in the range of 0.40–0.78, consistent with the high efficiency of intermittent searching8,32. Shorter and less-structured oligomers were more successful in achieving a FRET event. In particular, the 15bp system resulted in the most FRET events, with event fractions of 0.78 and 0.68 on hydrophobic and hydrophilic surfaces, respectively. The 30bp, overhang, and hairpin systems were less successful in achieving FRET events, with event fractions of 0.59–0.65 on the hydrophobic surface and 0.40–0.58 on the hydrophilic surface. Interestingly, event fractions were consistently higher on hydrophobic than on hydrophilic surfaces. This was the particularly dramatic for the hairpin system, which exhibited event fractions of 0.60 and 0.40 on hydrophobic and hydrophilic surfaces, respectively. These event fractions had no apparent correlation to the absolute nonspecific adsorption rate, which was primarily a function of the target oligonucleotide molecular weight (Figure S1).


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Fig 4. Fraction of trajectories that resulted in a FRET event, calculated by dividing the number of trajectories that engaged in FRET by the total number of trajectories. Error bars are Poisson standard errors.

Association Lifetime Distributions As described above, the lifetime of each FRET event was determined and these data were compiled in the form of complementary cumulative lifetime distributions, which were generally well-described by a bi-exponential fitting function. Since this analysis is central to the interpretations made below, a general description is given here. Figure 5 shows a generic biexponential distribution (plotted on semi-logarithmic axes), annotated with features that are related to the three independent fitting parameters.


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Fig 5. A generic bi-exponential complementary cumulative association lifetime distribution, showing how different independent parameters affect the shape of the distribution.

In the data shown below, the majority of association events were short in duration, leading to a rapid initial decay of the cumulative distribution. The initial slope is therefore inversely proportional to the characteristic lifetime, 1, of this short-lived population. The distributions eventually transition to a much shallower slope, and the behavior at long times is dominated by the long-lived population. Therefore, the asymptotic slope at long times is inversely proportional to the characteristic lifetime, 2, of the long-lived mode. The fraction of events associated with this long-lived population, f2, is indicated by the extrapolated y-intercept of the asymptotic part of the distribution as shown in Figure 5.

We will argue below that the short-lived mode (which is present even in experiments with noncomplementary strands) represents non-specific associations and/or partial hybridization, while the long-lived mode represents true stable hybridization into the maximal duplex state. Therefore, we will be particularly interested in f2 and 2, which give the fraction of events that


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lead to hybridization and the characteristic lifetime of hybridization, respectively. Indeed, as expected, 2 is systematically greater for DNA pairs with greater complementarity, as expected for hybridization lifetimes. Therefore, in the following, we will define the parameter f2 as the “success fraction”, denoting the fact that it represents the fraction of events that successfully enter the long-lived (i.e. hybridized) state.

In order to ensure that photobleaching or photoblinking did not affect the long-lived tail of the different distributions, we performed a control experiment for the 30bp system where the excitation power of the laser at the interface was varied from 30 mW to 50 mW. The shapes of the distributions were identical within experimental uncertainty over this range of laser power (Figure S2), and the fitted long-lived lifetime was similarly identical across the range of laser power (Table S2). Since photophysical phenomena are expected to be proportional to excitation intensity, the independence of measured lifetimes on intensity suggests that these data were not influenced by artifacts such as photobleaching or photoblinking.

Strand Lengths and Structure Affect Success Fraction and Secondary Lifetime

Figure 6 shows the complementary cumulative lifetime distributions for each of the systems studied on both hydrophobic and hydrophilic surfaces in 1X PBS buffer, and the parameters associated with the bi-exponential fits are given in Table 2. Since the trends identified here were similar at higher ionic strength (Table S1), we illustrate the behavior by focusing specifically on the results in 1X PBS.


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Despite the absence of complementarity between the control target strand and the immobilized probe, a small number of FRET events were still observed. The distributions of association times for the non-complementary control systems decay very quickly, consistent with a very short characteristic lifetime (10.26 s on a hydrophobic surface, 10.17 s on a hydrophilic surface). While the distribution for the control system exhibited an apparent “tail” on the hydrophobic surface, it was composed of a few long-lived FRET events leading to large uncertainties in the data and fitting parameters for the long-lived population. We speculate that these were actually a few anomalous events. In fact, when these data were decomposed using the Maximum Entropy Method, only a single peak was observed, suggesting that only one (very short) timescale was truly significant. One peak was also observed for the control system on the hydrophilic surface. Taken together, these observations suggest that some transient FRET events are the result of non-specific interactions between DNA strands that are independent of Watson-Crick base pairing. Obviously, such non-specific interactions can also occur between complementary strands, e.g. if the strands are misaligned.

Fig 6. Complementary cumulative association lifetime distributions for all systems in 1X PBS buffer, on (a) hydrophobic and (b) hydrophilic surfaces.


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Table 2. Short lifetimes, long-lived fractions, and long-lived lifetimes from biexponential fits as shown in Figure 6. Values in parentheses indicate uncertainty in the last digit. All experiments were performed in 1X PBS buffer.

Hydrophobic

Hydrophilic

𝜏1 (s)

f2

𝜏2 (s)

15bp

0.46(5)

0.06(1)

2.4(5)

30bp

0.3(1)

0.047(3)

3.8(5)

Overhang

0.32(3)

0.041(6)

1.7(1)

Hairpin

0.23(1)

0.034(1)

1.38(7)

Control

0.26(5)

0.01(6)

4(5)

15bp

0.33(4)

0.08(1)

2.4(4)

30bp

0.41(5)

0.11(2)

2.9(6)

Overhang

0.27(6)

0.04(2)

2.4(5)

Hairpin

0.17(9)

0.12(9)

1.0(9)

Control

0.17(2)

0.0(2)

0(6)

For oligonucleotides containing sequences complementary to the immobilized probes, the shortlived lifetimes determined from bi-exponential fits were in the range 0.17–0.46 s. Given the fitting uncertainties on these parameter values and the temporal resolution of the experiment (the capture time for each frame was 100 ms), the differences between these values were relatively insignificant. Thus, neither strand length nor surface chemistry appeared to alter the characteristic short-lived lifetime in a systematic way. Moreover, all of these short-lived lifetimes were similar in magnitude to lifetimes associated with non-complementary control strands, and many were statistically indistinguishable.

The long-lived lifetimes, τ2, obtained from bi-exponential fits were in the range of 1-4 s, and generally correlated with the degree of complementarity between probe and target. For example, on hydrophobic surfaces, the 15bp and 30bp systems had long-lived timescales of τ2=2.4 s and τ2=3.8 s, respectively. Interestingly, the overhang and hairpin systems, both of which had 15 base pairs when fully annealed, exhibited shorter long-lived lifetimes of 1.7 s and 1.38 s, respectively.


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While the control sequence had a large apparent τ2 value (4±5s), this was actually an artifact due to an extremely small number of anomalous events, which is underscored by the fact that the uncertainty in the parameter is actually greater than the value itself.

The same trends were generally observed on hydrophilic surfaces, where again the long-lived lifetimes correlated with the degree of complementarity between probe and target, and the hairpin system exhibited the shorted lifetime. Indeed, most of the τ2 values were actually equal within experimental uncertainty to those on hydrophobic surfaces. Interestingly, the exception to this involved the overhang system, where τ2 was significantly longer on hydrophilic surfaces compared to hydrophobic surfaces.

Most of the association events were short-lived; in fact, only 3–12% of FRET events were assigned to the long-lived mode of association. The most notable trend involved the dependence on surface chemistry, where successful long-lived associations were apparently hindered on hydrophobic surfaces (f2 in the range 0.03–0.06) and facilitated on hydrophilic surfaces (f2 in the range 0.04–0.12).

Salt Concentration Affects Successful Entry into the Long-Lived State Figure 7 shows the complementary cumulative lifetimes for the 15bp system, the 30bp system, and the hairpin system on a hydrophobic surface, at 1X, 5X, and 10X concentrations. Ionic strength significantly influenced the probability for long association times for the 15bp and 30bp systems (Figures 7A, 7B). The ionic strength had little effect on the association lifetimes for the


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hairpin system (Figure 7C), implying that the presence of target secondary structure masked any potential effect of ionic strength.

The success fraction was the parameter most affected by salt concentration (Figure 8), as opposed to the long-lived lifetime. The success fraction f2 for the 15bp system increased with increasing salt concentration from ~0.06 in 1X PBS buffer, to ~0.10 and ~0.15 in 5X and 10X PBS, respectively. Similarly, for the 30bp system, f2 increased from ~0.05 to ~0.06 to ~0.08 in 1X, 5X, and 10X PBS buffers, respectively. The hairpin system was not significantly affected by salt concentration, with the success fraction only fluctuating slightly in the range 0.031-0.037 for the entire range of ionic strength.

The characteristic timescales associated with the long-lived states, τ2, were relatively insensitive to salt concentration (Figure S3). In particular, 𝜏2 ≈ 2 s and 𝜏2 ≈ 3.5 s for the 15bp and 30bp systems, respectively, regardless of ionic strength. The secondary lifetime for the hairpin system shifted from ~1.38 s at 1X PBS to ~1.26 s and ~1.07 s at 5X PBS and 10X PBS, respectively.


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Fig 7. Complementary cumulative association lifetime distributions for (A) the 15bp system, (B) the 30bp system, and (C) the hairpin system on a hydrophobic surface, indicating how they varied with salt concentration.


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Fig 8. The change in f2 on a hydrophobic surface as a result of PBS concentration for (A) the 15bp system, (B), the 30bp system, (C), the hairpin system.


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Discussion Frequency of FRET events Notably, the largest effect on the event fraction fe was the presence or absence of complementarity, since the control strands exhibited much lower values than experiments with complementarity. While random collisions between non-complementary target probe pairs were expected to occur at approximately the same frequency as between complementary pairs, the former collisions were apparently much less likely to lead to a resolved FRET event. The likely reason for this is related to the temporal resolution of the experiments, i.e. association events that are long enough to resolve are more likely to result from some degree of partial hybridization (i.e. out-of-sequence pairing), as opposed to aromatic stacking and hydrophobic interactions alone.

Among complementary sequences, the largest event fractions occurred with the 15bp system, which had event fractions of 0.78 and 0.68 on hydrophobic and hydrophilic surfaces, respectively, whereas systems with longer and/or more structured strands exhibited lower event fractions in the range 0.4-0.65. If out-of-sequence pairing is required to produce resolvable FRET events, as hypothesized above, then it is sensible that shorter strands were more likely to randomly collide in a configuration where this can occur, because they have a lower probability


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space of complementary sequences; therefore a collision was more likely to result in the association of two or more in-sequence pairs.

Notably, event fractions were systematically higher on the hydrophobic surface than on the hydrophilic surface. This was probably due in part to the fact that the DNA targets were significantly more mobile on hydrophobic surfaces, and therefore more likely to encounter an immobilized probe prior to desorption33. There was no apparent effect of salt concentration on event fractions for any system.

Hybridization and non-specific interactions In the experiments described above, FRET events resulted from both non-specific and specific (i.e. hybridization) interactions between immobilized and mobile DNA oligomers. For example, even non-complementary (“control”) oligomers exhibited short FRET events, with characteristic time-scales on the order of a few hundred ms. Interestingly, when the FRET events between complementary strands were decomposed, a similar short-lived timescale was again observed (along with a long-lived time-scale on the order of seconds), suggesting that these complementary oligomers could also engage in non-specific interactions. However, complementary oligomers also exhibited a second longer-lived mode of association with a characteristic timescale on the order of seconds, and this longer timescale increased with a larger number of complementary base pairs. These observations suggest that the longer-lived associations represented actual hybridization events. In this scenario, the short-lived lifetime represents brief associations between the target and probe, possibly involving aromatic-stacking


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interactions or out-of-sequence base pairing, that were sufficiently long to create a measurable FRET event but did not proceed to full hybridization. Interestingly, while the observable shortlived associations had similar timescales for complementary and non-complementary pairs, they were much more frequent for complementary oligomers, which could engage in both aromatic stacking and partial hybridization, than for non-complementary DNA pairs, which could engage only in completely non-specific interactions such as aromatic stacking.

Factors influencing hybridization Many interrelated factors influenced the likelihood of hybridization, which was parameterized by the success fraction, f2. Generally speaking, shorter and less-structured target oligonucleotides were more successful at hybridizing to immobilized probes. For example, on hydrophobic surfaces, where interactions with the DNA backbone were weak33, f2 was larger for the 15bp system than the 30bp, hairpin, and overhang systems, which had longer and/or structured targets. We speculate that longer oligonucleotides had a lower probability of interacting in a way that would lead to hybridization, i.e. two interacting bases in a given nonspecific associated state were less likely to be complementary for longer DNA strands, due to the larger “searching space” that must be explored before nucleating an in-sequence dsDNA base pair leading to hybridization. Moreover, since longer oligonucleotides are expected to adopt conformations that sequester hydrophobic nucleobases33 (particularly on hydrophobic surfaces), these bases are less available to engage in hybridization interactions. A similar effect is expected to be relevant for the hairpin system, where target bases are strongly interacting with other target bases, and therefore less available to hybridize with probe bases, which has previously been shown to inhibit hybridization34.


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Interestingly, on the hydrophilic surface, which interacts more strongly with the DNA backbone leading to extended (as opposed to compact) DNA conformations33,35,36, targets were more likely to hybridize than on the hydrophobic surface. We speculate that the extended DNA conformations resulted in greater base exposure, increasing the likelihood that target and probe will collide in productive ways. Electrostatic effects also influenced the likelihood of hybridization, this was particularly apparent from ionic strength effects where increased electrostatic screening increased the probability of long-lived events for both the 15bp and 30bp systems. In fact, electrostatic repulsion may also contribute to the decreased hybridization probability of longer strands discussed above, since longer DNA molecules will necessarily have a greater negative charge.

The results also suggested that the lifetime of the hybridized state could be affected by competitive interactions. Notably, hybridization lifetimes were significantly shorter for the hairpin system than for any other system despite the fact that the degree of complementarity was the same as for the 15pb and overhang systems. For the hairpin system, we speculate that the partially melted state can be stabilized by self-hybridization, essentially leading to a branch migration mechanism similar to what occurs in DNA strand displacement reactions37,38.

Hybridization efficiency In contrast with ensemble-averaging experiments, which measure only an overall hybridized amount or fraction, the single molecule results described above deconvolute this fraction into several independent parameters. In particular, we calculated


28

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 the fraction of adsorbed target molecules resulting in a FRET event, i.e. the “event fraction” fe.  the fraction of FRET events that resulted in hybridization, i.e. the “success fraction” f2.  the hybridized state lifetime 𝜏2 .

Each of these parameters represents a physically distinct mechanism and may be influenced in different ways by environmental conditions. For example, while event fractions were uniformly smaller on hydrophilic compared to hydrophobic surfaces, success fractions generally had the opposite trend. Therefore, it is interesting to consider how the three parameters interact to create an overall efficiency of hybridization. As shown in Table 3, this can be represented by a combined parameter 𝜏ℎ = 𝑓𝑒 𝑓2 𝜏2 ̅̅̅

(3)

which represents the long-lived lifetime weighted by the event fraction as well as the secondary lifetime (success) fraction. Conceptually, it represents the mean amount of time a given adsorbed object will be in a hybridized state.


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Table 3. Event fraction, success fraction, long-lived lifetime, and mean long-lived lifetime as a function of strand structure and surface chemistry. Values in parentheses indicate uncertainty in the last digit, except for the mean long-lived lifetime for the control, where the error exceeded the lifetime value. All experiments were performed at 1X PBS concentration Surface 15bp

30bp

Overhang

Hairpin

Control

𝑓𝑒

𝑓2

𝜏2 (s)

𝜏ℎ (s) ̅̅̅

Hydrophobic

0.78

0.06(1)

2.4(5)

0.11(4)

Hydrophilic

0.68

0.08(1)

2.4(4)

0.14(3)

Hydrophobic

0.59

0.047(3)

3.8(5)

0.10(1)

Hydrophilic

0.51

0.11(2)

2.9(4)

0.16(4)

Hydrophobic

0.65

0.041(6)

1.7(1)

0.046(8)

Hydrophilic

0.58

0.04(2)

2.4(5)

0.06(3)

Hydrophobic

0.60

0.034(1)

1.38(7)

0.028(2)

Hydrophilic

0.40

0.12(9)

1.0(9)

0.05(6)

Hydrophobic

0.06

0.01(6)

4(5)

0.00(1)

Hydrophilic

0.02

0.0(2)

0(5)

0.000(5)

Notably, despite the fact that event fractions were generally higher on the hydrophobic surface than the hydrophilic surface, the mean long-lived lifetimes, ̅̅̅, 𝜏ℎ were still longer on the hydrophilic surface due to larger values of f2, τ2, or both. The 15bp and 30bp systems had the longest mean long-lived lifetimes, with overhang and hairpin systems having values of ̅̅̅ 𝜏ℎ that were a factor of 2–3 smaller. Because 𝑓𝑒 did not appear to change significantly as a function of salt concentration, the changes in ̅̅̅ 𝜏ℎ with ionic strength were primarily due to the changes in success fraction or long-lived lifetime (Table S3).Interestingly, this dramatic overall reduction of hybridization efficiency for the overhang and hairpin system was not ascribed to a single parameter, but instead to a synergistic combination of inefficient search and hybridization, and short hybridization times.


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Summary and Conclusions DNA hybridization events investigated using single-molecule FRET techniques have shown that association and dissociation of DNA at solid-liquid interfaces exhibits complex behavior that is not reducible to a single type of binding and/or melting event. This work has focused on three distinct kinetic parameters, which were described as the “event fraction” (related to searching success), the “success fraction” (related to the relative likelihood of hybridization to non-specific association), and the characteristic hybridization lifetime, and the physical mechanisms behind these behaviors were investigated as a function of oligonucleotide structure, ionic strength, and surface chemistry. As expected, more thermodynamically stable DNA duplexes exhibited longer hybridization times; however, the long, stable hybridization chains weren’t necessarily the most likely to enter into this stable state. In general, shorter and less-structured target strands were more likely to associate with immobilized probe strands, and to hybridize with the probes once associated. While “searching” was very efficient on hydrophobic surfaces, this effect was largely overwhelmed by the fact the hybridization was more likely and stable on hydrophilic surfaces. Electrostatic screening mainly influenced the probability of hybridization, i.e. electrostatic repulsion decreased the likelihood of hybridization compared to non-specific associations. This detailed picture of the dynamics of surface-mediated hybridization will provide guidance in choosing design parameters for applications that require rapid, efficient, selective, and/or stable hybridization at interfaces.

In this work, DNA association/dissociation at very fast timescales was limited by experimental temporal resolution, which was essentially limited by photon counts and shot noise. While this resolution can be improved by using higher excitation intensity, this would be at the expense of


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photobleaching, which would limit our ability to measure long hybridization times. Here, we optimized conditions to measure long-lived events so that we could accurately decompose the association distributions in order to extract the fraction and timescale associated with hybridization. Therefore, association lifetimes shorter than 200ms were removed from further analysis. In principle, higher excitation intensity could be used to perform separate experiments to investigate the subtle dynamics of shorter associations, i.e. to investigate the possibility of multiple intermediate association states.


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References (1)

Levicky, R.; Horgan, A. Physicochemical perspectives on DNA microarray and biosensor technologies. TRENDS Biotechnol. 2005, 23 (3), 143–149.

(2)

Ventimiglia, G.; Alessi, E.; Petralia, S. A bridge-like solution for Universal Microarray applications. Sensors Actuators B. Chem. 2016, 232, 102–106.

(3)

Hacia, J. G.; Brody, L. C.; Chee, M. S.; Fodor, S. P. A.; Collins, F. S. Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and two-colour fluorescence analysis. Nat. Genet. 1996, 14, 441–447.

(4)

Cutler, D. J.; Zwick, M. E.; Carrasquillo, M. M.; Yohn, C. T.; Tobin, K. P.; Kashuk, C.; Mathews, D. J.; Shah, N. A.; Eichler, E. E.; Warrington, J. A.; Chakravarti, A. High-Throughput Variation Detection and Genotyping Using Microarrays. Genome Res. 2001, 11, 1913–1925.

(5)

Peterson, E. M.; Manhart, M. W.; Harris, J. M. Competitive Assays of LabelFree DNA Hybridization with Single- Molecule Fluorescence Imaging Detection. Anal. Chem. 2016, 88, 6410–6417.

(6)

Chen, Y.; Nakamoto, K.; Niwa, O.; Corn, R. On-Chip Synthesis of RNA Aptamer Microarrays for Multiplexed Protein Biosensing with SPR Imaging Measurements. Langmuir 2013, 28 (22), 8281–8285.

(7)

Skaug, M. J.; Mabry, J. N.; Schwartz, D. K. Single-Molecule Tracking of


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Polymer Surface Diffusion. J. Am. Chem. Soc. 2013, 136 (4), 1327–1332. (8)

Monserud, J. H.; Schwartz, D. K. Mechanisms of Surface-Mediated DNA Hybridization Mechanisms of Surface-Mediated DNA Hybridization. ACS Nano 2014, 8 (5), 4488–4499.

(9)

Yakovchuk, P.; Protozanova, E.; Frank-kamenetskii, M. D. Base-stacking and base-pairing contributions into thermal stability of the DNA double helix. Nucleic Acids Res. 2006, 34 (2), 564–574.

(10) Swart, M.; van der Wijst, T.; Fonseca Guerra, C.; Bickelhaupt, F. M. Π-Π Stacking Tackled With Density Functional Theory. J. Mol. Model. 2007, 13 (12), 1245–1257. (11) Ouldridge, T. E.; Sulc, P.; Romano, F.; Doye, J. P. K.; Louis, A. A. DNA hybridization kinetics: zippering, internal displacement and sequence dependence 1. Nucleic Acids Res. 2013, 41, 8886–8895. (12) Sambriski, E. J.; Schwartz, D. C.; Pablo, J. J. De. Uncovering pathways in DNA oligonucleotide hybridization via transition state analysis. Proc. Nat. Acad. Sci. USA 2009, 106 (43), 18125–18130. (13) Frank-kamenetskii, M. D.; Prakash, S. Fluctuations in the DNA double helix : A critical review. Phys. Life Rev. 2014, 11 (2), 153–170. (14) Manghi, M.; Destainville, N. Physics of base-pairing dynamics in DNA. Phys. Rep. 2016, 631, 1–41.


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(15) Kastantin, M.; Schwartz, D. K. DNA hairpin stabilization on a hydrophobic surface. Small 2013, 9 (6), 933–941. (16) Moerner, W. E.; Fromm, D. P. Methods of single-molecule fluorescence spectroscopy and microscopy Methods of single-molecule fluorescence spectroscopy and microscopy. 2007, 3597 (2003). (17) Honciuc, A.; Howard, A. L.; Schwartz, D. K. Single molecule observations of fatty acid adsorption at the silica/water interface: Activation energy of attachment. J. Phys. Chem. C 2009, 113 (6), 2078–2081. (18) Peterson, E. M.; Manhart, M. W.; Harris, J. M. Single-Molecule Fluorescence Imaging of Interfacial DNA Hybridization Kinetics at Selective Capture Surfaces. Anal. Chem. 2016, 88, 1345–1354. (19) Leszczynski, J.; Hobza, P.; Heyrovsky, J. Electronic Properties, Hydrogen Bonding, Stacking, and Cation Binding of DNA and RNA Bases. Biopolymers 2002, 61, 3–31. (20) Liu, C.; Obliosca, J. M.; Liu, Y.-L.; Chen, Y.-A.; Jiang, N.; Yeh, H.-C. 3D single-molecule tracking enables direct hybridization kinetics measurement in solution. Nanoscale 2017, 9 (17), 5664–5670. (21) Chen, J.; Poddar, N. K.; Tauzin, L. J.; Cooper, D.; Kolomeisky, A. B.; Landes, C. F. Single-Molecule FRET Studies of HIV TAR − DNA Hairpin Unfolding Dynamics. J. Phys. Chem. B 2014, 118, 12130–12139.


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(22) Sobek, J.; Schlapbach, R. Minimizing DNA microarrays to a single molecule per spot: using zero-mode waveguide technology to obtain kinetic data for a large number of short oligonucleotide hybridization reactions. 2017. (23) Johnson-buck, A.; Walter, N. G. Discovering anomalous hybridization kinetics on DNA nanostructures using single-molecule fluorescence microscopy. Methods 2014, 67, 177–184. (24) Dupuis, N. F.; Holmstrom, E. D.; Nesbitt, D. J. Single-Molecule Kinetics Reveal Cation-Promoted DNA Duplex Formation Through Ordering of Single-Stranded Helices. Biophysj 2013, 105 (3), 756–766. (25) Tsukanov, R.; Tomov, T. E.; Masoud, R.; Drory, H.; Plavner, N.; Liber, M.; Nir, E.; Sheva, B. Detailed Study of DNA Hairpin Dynamics Using SingleMolecule Fluorescence Assisted by DNA Origami. J. Phys. Chem. B 2013, 117 (40), 11932–11942. (26) Monserud, J. H.; Macri, K. M.; Schwartz, D. K. Toehold-Mediated Displacement of an Adenosine-Binding Aptamer from a DNA Duplex by its Ligand. Angew. Chem. Int. Ed. 2016, 55, 13710–13713. (27) Kastantin, M.; Schwartz, D. K. Connecting Rare DNA Conformations Molecule Resonance Energy Transfer. ACS Nano 2011, 5 (12), 9861–9869. (28) Walder, R.; Schwartz, D. K. Single molecule observations of multiple protein populations at the oil-water interface. Langmuir 2010, 26 (16), 13364–13367.


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(29) McLoughlin, S. Y.; Kastantin, M.; Schwartz, D. K.; Kaar, J. L. Singlemolecule resolution of protein structure and interfacial dynamics on biomaterial surfaces. Proc. Nat. Acad. Sci. USA 2013, 110 (48), 19396– 19401. (30) Steinbach, P. J.; Ionescu, R.; Matthews, C. R. Analysis of Kinetics Using a Hybrid Maximum-Entropy/Nonlinear-Least-Squares Method: Application to Protein Folding. Biophys. J. 2002, 82 (4), 2244–2255. (31) Steinbach, P. J. Filtering artifacts from lifetime distributions when maximizing entropy using a bootstrapped model. Anal Biochem. 2012, 427 (1), 102–105. (32) Monserud, J. H.; Schwartz, D. K. Interfacial Molecular Searching Using Forager Dynamics. Phys. Rev. Lett. 2016, 116, 98303. (33) Monserud, J. H.; Schwartz, D. K. Effects of Molecular Size and Surface Hydrophobicity on Oligonucleotide Interfacial Dynamics. Biomacromolecules 2012, 13 (12), 4002–4011. (34) Gao, Y.; Wolf, L. K.; Georgiadis, R. M. Secondary structure effects on DNA hybridization kinetics: A solution versus surface comparison. Nucleic Acids Res. 2006, 34 (11), 3370–3377. (35) Cherny, D. I.; Fourcade, A.; Svinarchuk, F.; Nielsen, P. E.; Malvy, C.; Delain, E. Analysis of various sequence-specific triplexes by electron and


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atomic force microscopies. Biophys. J. 1998, 74 (2 Pt 1), 1015–1023. (36) Martínez, J. M.; Elmroth, S. K. C.; Kloo, L. Influence of Sodium Ions on the Dynamics and Structure of Single-Stranded DNA Oligomers: A Molecular Dynamics Study. J. Am. Chem. Soc. 2001, 123 (49), 12279–12289. (37) Wang, L.; Fang, L.; Liu, S. Responsive hairpin DNA aptamer switch to program the strand displacement reaction for the enhanced electrochemical assay of ATP. Analyst 2015, 140 (17), 5877–5880. (38) Lilley, D. M. J. The Structure Of the Four-Way Junction in DNA. Annu. Rev. Biophys. Biomol. Struct. 1993, 22, 199–328.

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Surface-Mediated DNA Hybridization: Effects of DNA Conformation

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